A generic device for multispot scanning microscopy is described for example in WO-13 131 808 A1 and has the following components: a multicolour light source for providing at least one illumination light beam, a splitting device for splitting the illumination light beam into a plurality of illumination sub-beams, first optical means for providing an illumination optical path to guide and focus the individual illumination sub-beams respectively into a light spot on or in a specimen to be examined, a scan unit for guiding the light spots over the specimen, a detection unit for detecting detection light radiated by the specimen in detection sub-beams after irradiation with the individual illumination sub-beams, second optical means for providing a detection optical path to guide the detection sub-beams to the detector unit and a control and evaluation unit for controlling the scan unit and for evaluating the detection light detected by the detection unit.
A generic method for multispot scanning microscopy is also disclosed in WO 13-131 808 A1. Here, the following steps are carried out: at least one illumination light beam is provided with a multicolour light source, the illumination light beam is split into a plurality of illumination sub-beams, the individual illumination sub-beams are guided in an illumination optical path respectively into a light spot on or in a specimen to be examined and scanned over this, detection light radiated by the specimen in detection sub-beams after irradiation with the individual illumination sub-beams is guided to a detector unit and detected by this.
Laser scanning microscopy has developed into an indispensable tool in the life sciences. In particular the imaging of three-dimensional structures in a scattering background additionally makes the method suitable for a broad range of biological-medical applications. In particular the multifaceted nature of the method has led to an extensive dissemination of corresponding systems and a broad field of application.
However, the method continues to have a series of problems. These include firstly a significant tendency for bleaching of the fluorescence and generally for photo damage to the specimen, secondly a relatively slow image recording and thirdly increased noise in comparison with wide field methods.
The reasons for these issues lie in the type of image formation. This is generally carried out so that a point, more specifically the volume of the illumination point spread function (PSF), sequentially raster-scans or scans the specimen. The out-of-focus light is discriminated with respect to the focal signal light at a pinhole. This leads to an image production with the property of optical sectioning. This means that only the light from the focal plane contributes to the signal. In this way a blur-free imaging also of optically denser and slightly scattering specimens is possible. The scanning of the specimen with a laser beam leads on the one hand to high powers in a focal spot. This is the illumination spot, onto which or into which the illumination light is focused on or in a specimen. On the other hand the scanning of the specimen facilitates only a slow image formation which is limited by the speed of the scanners or by the emission rate of the dyes in the specimen. The emission rate of the dyes in the specimen is by nature low because only small volumes are respectively scanned in the specimen.
Irrespectively of its very wide dissemination in the life sciences, confocal microscopy has in particular the problem that for signal generation with an acceptable signal-to-noise ratio (SNR) a certain number of photons must be generated in a short time (e.g. 10 MHz rate signal photons for detection of 10 photons in a pixel time of 1 μs with a SNR of approximately 3; the rate in the specimen must still be significantly higher due to the losses in the system). This is associated firstly with the bleaching of the specimen and secondly with damage that makes the examination of many parameters in the field of living cell microscopy impossible or at least considerably more difficult.
Shortening the pixel time leads to a somewhat faster image formation, but on the other hand to even higher powers in the focal spot for sufficiently high signal generation. The inherent dilemma in relation to the three basic requirements: 1) image speed, 2) good signal-to-noise ratio and 3) low photo damage cannot therefore be resolved with the single spot laser scanning microscope.
A further important property of fluorescence microscopy is the spectral wideband thereof. The excitation maximums of the different dyes lie in a range extending from UV via the visible spectrum as far as the infrared spectral range. In standard commercial systems, excitation in the wavelength range of 400 nm-645 nm or higher is generally available. The scanning of the specimen with only one laser focus is also disadvantageous in relation to this point. A plurality of excitation wavelengths can be focussed in the same focal spot and therefore a plurality of fluorophores can be simultaneously excited. However, even in the case of a spectrally selective detection, so called cross-excitations always arise and detection of undesired spectral portions which can lead to a false assignment of structures or an undesired background.
Somewhat better conditions with respect to the achievable SNR are offered by a spinning disk (SD) system. Here, the light is distributed onto many (approximately 1000) focal volumes. With approximately the same SNR, the illumination time of a certain region in the specimen thus increases with simultaneously lower intensity per focal volume. This leads overall to behaviour that is greatly improved with respect to photo damage through phototoxicity. However, these systems cannot be used flexibly. In particular the spot distance cannot be varied. In addition the detection is limited to cameras, which makes a detection of more than 2 spectral channels considerably more difficult. In addition it is not possible to electronically zoom into the specimen with these non-scanner-based systems. Point measurements, such as fluorescence correlation spectroscopy, are not possible at all with these systems.
Further multipoint systems have been described in DE 102 15 162 B4, WO 13 131 808 A1, U.S. Pat. No. 6,028,306 A. A problem with these systems always arises in the provision of the scanning laser points. Arrangements for this are described for example in DE 102 15 162 B4 and DE 10 2010 047 353 A1.
A property of all these systems is additionally that they offer only a passive multibeam generation. This means that, for example, the colour splitting is generally fixed and is generally equal for all beams. In AT-131942-E, different lasers are used for different scanning points, which greatly limits the flexibility of this system. Furthermore the brightnesses of individual beams and colours cannot be individually tuned, which constitutes a problem when structures with a differing level of marking with different dyes are to be examined.
In measurements where high quality in particular is the key factor, a temporal multitrack is generally recorded. Here, the images of different fluorophores, which each require a certain wavelength for excitation and a certain spectral configuration for detection, are recorded one after the other in time, although the conventional systems in principle make simultaneous recording possible. Even if the user has a time disadvantage here, the benefit with respect to the quality achieved outweighs this.
AT-131942-E indicates a system which avoids the effect of spectral crosstalk by the spectral excitation taking place at respectively different locations in the specimen. Reference is made here to a spatial spectral multitracking. However, the colour channels are thereby fixedly predefined and not configurable. This system cannot therefore be optimised, in the case of specimens that contain fewer than the number of dyes that can be detected in principle, with respect to other parameters such as for example specimen preservation or speed, as the wavelength distribution is fixedly predefined.